With a growing world economy and request for higher living standard, the demand for fragrance is on the rise. However, supplies of animal or plant based fragrance are limited, due to restraints in natural resources and animal protection. Attempts have been made to produce fragrances or their precursors from renewable sources such as microorganisms.
Ambergris is a prized, traditional fragrance ingredient that is a byproduct of the whale intestine. Ambrox, a substitute for ambergris, is produced by a chemical conversion of the diterpene sclareol, which is currently obtained from clary sage. Ambrox can also be generated from the related diterpene abienol, which has been found in fir and tobacco (Barrero et al. 1993, Tetrahedron 49:10405-10412). Also see FIG. 1.
The pathways to both abienol and sclareol in plants are proposed to involve two steps. The first step consists of the conversion of the isoprenoid pathway molecule geranylgeranyl diphosphate (GGPP) to a common intermediate named labda-13-en-8-ol diphosphate (LDPP) through the activity of a class II diterpene synthase (diTPS). The second step is catalyzed by a class I diTPS. There are several type of class I diTPS, each responsible for producing a specific end product. For example, abienol synthase (ABS) is for abienol production, and sclareol synthase (Scs) is responsible for sclareol production. See FIG. 2.
The enzymes involved in the two-step conversion of GGPP to sclareol or GGPP to abienol are plant specific and can be in the form of two independent enzymes or a single enzyme with two active sites. For example, in abienol production by tobacco (Sallaud et al. 2012, Plant J., 72(1):1-17), the class II diTPS of tobacco (referred to as NtCPS2 by Sallaud et al., and referred to as Nt-class II-diTPS by the present disclosure) and the class I diTPS synthase of tobacco (referred to as abienol synthase of tobacco or Nt-ABS by the present disclosure), are in the form of two different protein molecules. Similarly, in sclareol production by clary sage (Schalk et al. 2012, Journal of Am. Chem Soc. 134:18900-18903); (Caniard et al. 2012, BMC Plant Biology 12:119), the class II diTPS of clary sage (referred to as Ss LPS by Schalk et al, and referred to as Ss-class II-diTPS by the present disclosure) and the class I diTPS synthase of clary sage (referred to as sclareol synthase of clary sage or Ss-Scs by the present disclosure), are also in the form of two independent protein molecules. In contrast, in the production of abienol by fir (Zerbe et al. 2012, J. Biol. Chem. 287:12121-12131), both class I and class II diTPS subunits reside on one bifunctional class I/II abienol synthase (referred to as AbCAS by Zerbe et al. and by the present disclosure). Therefore in fir, GGPP is converted to abienol in the presence of the single bifunctional class VII abienol synthase. See FIG. 2.
Plant sources for sclareol and abienol are considered to be unreliable; thus a process for microbial production of either product could have commercial value. The class II diTPS and sclareol synthase genes from clary sage have been isolated and simultaneously expressed in E. coli, resulting in titers of approximately 1.5 grams per liter of sclareol in lab scale fermenters. Measurable production in E. coli of abienol has been achieved by the simultaneous expression of the class II diTPS and abienol synthase genes of tobacco, or the expression of the individual class VII abienol synthase of fir. However, production of abienol based on the existing methods is very low. It could therefore be desirable to produce abienol at a much higher titer.